It has been widely attempted to produce a protein that can be used as a pharmaceutical and a reagent via gene recombination technology. Preferably, in view of the ease of handling and efficiency, microorganisms such as E. coli, Bacillus subtilis, molds, and yeasts, insects such as silkworms, mammalian animals such as bovines, and culturable plant, insect, and animal cells have recently been used in gene recombination technology. A method of producing a protein using gene recombination technology has been widely used. However, the use of such method results in, for example, a low expression level for a protein of interest, the expression of a proteins having no activity, and formation of aggregates, which have been problematic. Thus, it is necessary to carry out a trial-and-error process such as examination of culture conditions, growth conditions, or induction conditions, or testing of various types of expression systems. Even after examination of these various conditions, many proteins that are difficult to produce have been reported.
Meanwhile, a protein synthesis method that does not use any such organisms or cells, called “cell-free protein synthesis,” has been known. Such cell-free protein synthesizing system is also described as an “in vitro transcription/translation system.” With this system, a template gene is subjected to transcription/translation using an extract or a crude fraction thereof prepared from E. coli, rabbit reticulocytes, wheat germ cells, or the like, resulting in protein synthesis. A cell-free protein synthesizing system is characterized in that limitations associated with the use of organisms and cells can be overcome. This is because, with the use of such system, there is a high probability that a protein that disturbs functions of organisms and cells can be synthesized, various types of proteins can be synthesized using a 96-well or 384-well format, and a variety of synthesis reaction conditions can be simultaneously examined.
However, it has been known that, even with the use of such cell-free protein synthesizing system, synthesized proteins form aggregates and do not constitute the relevant original structures, which have been problematic. For that reason, it is considered that proteins having intramolecular and/or intermolecular disulfide bonds are unable to properly crosslink between disulfide bonds. There are proteins having intramolecular and/or intermolecular disulfide bonds and those not having the same. Many of proteins that are transported and secreted onto the cellular surface or into the extracellular environment have disulfide bonds. Such proteins are believed to have particularly useful application. For instance, most of protein preparations such as insulin, cytokine, and blood cell growth factors that have been commercially available have a protein having intramolecular disulfide bonds as a component.
Thus far, in order to produce such proteins having intramolecular/intermolecular disulfide bonds with good efficiency, cell-free protein synthesizing systems have been improved. For instance, the following methods have been carried out: a method of adding microsome fractions to a cell extract (Non-Patent Document 1: Biochem. J. 254:805-810 (1988), hereafter referred to as Prior art 1); and a method of dialyzing a cell extract, a method of adding oxidized glutathione and reduced glutathione, a method of gel filtration, or a method of regulating oxidation-reduction (redox) potential (Non-Patent Document 2: FEBS Lett. 514:290-4 (2002); Non-Patent Document 3: Nature Biotech. 15:79-84 (1997); Patent Document 1: JP Patent Publication (Kokai) No. 2003-116590; Patent Document 2: WO 03/072796 A1, hereafter referred to as Prior art 2).
Also, a method of disulfide bond formation using a deletion variant of an enzyme that maintains a reduced state has been known (Proc. Natl. Acad. Sci. 96: 13703-13708 (1999), hereafter referred to as Prior art 3). With this method, a protein of interest can be expressed using E. coli lacking thioredoxin reductase and glutathion reductase. However, in general, such an enzyme-deficient cell line grows very slowly or requires specific culture conditions, which have been significant obstacles in terms of industrial availability. Such method employs a system that causes gene recombination to be carried out in E. coli such that proteins are expressed. However, as described above, there have been various limitations associated with the direct use of organisms. In addition, many organisms contain many unidentified enzymes and substrates controlling oxidation-reduction in addition to thioredoxin reductase and glutathion reductase (Non-Patent Document 4: Nature Review Molecular Cell Biology 3: 836-847 (2002)). Thus, even if a foreign gene can be expressed using such a deletion variant or protein synthesis can be carried out in a cell-free protein synthesizing system using a cell extract of a deletion variant, stable disulfide bond formation has been considered to be difficult. Therefore, a method of treating a cell extract with iodacetamide so as to inactivate such enzymes and substrates has been known (U.S. Pat. No. 6,548,276 B2, hereafter referred to as Prior art 4). In accordance with this method, it is possible to inactivate not only glutathion reductase and thioredoxin reductase but also many enzymes and substrates regulating intracellular oxidation-reduction. However, since iodacetamide modifies thiol in a nonspecific manner, it also modifies factors and enzyme groups related to transcription/translation, ribosomal protein, and the like. It is considered that efficiency or accuracy of an in vitro protein synthesis reaction deteriorates as a result, which is problematic.
Meanwhile, in accordance with a method that has been widely used, proteins obtained via recombinant production using organisms and cells, chemically synthesized peptides, or proteins synthesized via a cell-free protein synthesizing system are completely denatured using a denaturant, followed by regeneration thereof (Non-Patent Document 5: Biochemistry 26:3129-3134 (1987), hereafter referred to as Prior art 5). In the case of Prior art 5, since proteins are chemically synthesized or recovered as inactive insoluble matter when using organisms, toxic proteins can be produced, for example. Upon protein regeneration, cells are not used and reagents and salts freely combine with each other so that a proper disulfide bond can be introduced into a protein having disulfide bonds. When carrying out this method, however, separate steps of protein synthesis and protein structure regeneration are required, and protein structure regeneration is a time-consuming step (taking several days to one week), which have been problematic.
Further, in accordance with a method of measuring activity of an enzyme catalyzing promotion and/or isomerization of disulfide bonds that has been widely known, ribonuclease A (RNaseA) serving as a substrate is reduced so as to be denatured, RNaseA is refolded together with an enzyme, the activity of which is measured, so as to be regenerated, and the activity of RNase obtained is determined to be an index (Non-Patent Document 6: Biochem J. 1976 159:377-384).    Patent Document 1: JP Patent Publication (Kokai) No. 2003-116590 A    Patent Document 2: WO 03/072796 A1    Patent Document 3: U.S. Pat. No. 6,548,276 B2    Non-Patent Document 1: Biochem. J. 254:805-810 (1988)    Non-Patent Document 2: FEBS Lett. 514:290-294 (2002)    Non-Patent Document 3: Nature Biotech. 15:79-84 (1997)    Non-Patent Document 4: Nature Review Molecular Cell Biology 3:836-847 (2002)    Non-Patent Document 5: Biochemistry 26:3129-3134 (1987)    Non-Patent Document 6: Biochem J. 159:377-384 (1976)